Human embryonic stem cells (hESCs) hold tremendous potential for tissue engineering and regenerative medicine applications because of their unique combination of two properties, pluripotency and the capacity for infinite self-renewal. Thus, hESCs may serve as a safe, limitless supply of desired cells for cell-based therapies. To realize this potential, hESC researchers require culture systems that permit robust expansion of undifferentiated hESCs, provide efficient storage and recovery of hESCs, and direct differentiation of hESCs along desired developmental lineages. Design of such culture systems requires an understanding of how a variety of microenvironmental signals affect hESC growth and differentiation. These signals include spatial and temporal presentation of soluble proteins and other chemical factors, immobilized extracellular matrix components, mechanical stimuli, and cell-cell contact. In recent work we and others have demonstrated that constructing microenvironments that confine a variety of cell types, including hESCs, to 2-dimensional patterns or to 3-dimensional wells can have dramatic effects on intracellular signal transduction pathways, gene expression, and cell phenotype, including survival, proliferation, and differentiation. We have developed a method to confine hESCs to polymer microwells, constructed by soft lithography, by treating the surfaces between the microwells with a cell and protein repulsive coating and adsorbing extracellular matrix proteins to the surfaces inside the wells. As colonies fill the wells, each colony in the culture has the same size and shape as portions of the colony that grow outside the well are removed by fluid shear. Thus, these microwells serve as a valuable tool to assess the effects of colony morphology on hESC growth and differentiation. We have assembled a research team with expertise in materials science, hESC cell biology, cell physiology, and cell engineering to address how microwell confinement can be used in conjunction with other microenvironmental stimuli to design hESC culture systems. Our experiments will utilize hESC cell lines WA01 and WA09 from the NIH hESC Stem Cell Registry. In this study, we propose the following aims to develop a microwell-based culture system that (1) facilitates high density culture of undifferentiated hESCs, (2) provides efficient recovery of viable, undifferentiated hESCs following cryopreservation, and (3) directs differentiation of hESCs along desired developmental lineages: 1. Assess effects of hESC colony confinement on growth and differentiation in defined medium 2. Determine impact of colony confinement on recovery of undifferentiated hESCs following cryopreservation 3. Quantify optimum embryoid body size for generation of hESC-derived cardiac myocytes 4. Develop mechanically-compliant microwells for application of mechanical strain to confined hESC colonies